Validated HPLC analytical method with programmed wavelength UV detection for simultaneous determination of DRF-4367 and Phenol red in rat in situ intestinal perfusion study

A simple, precise and accurate isocratic reverse-phase liquid chromatography method with programmed wavelength detection has been validated to quantify DRF-4367 and Phenol red, simultaneously for application in rat in situ single pass intestinal perfusion study to assess intestinal permeability of DRF-4367, a novel cox-2 inhibitor. The method was validated on RP C-18 analytical column. Mobile phase consisted of sodium dihydrogen orthophosphate (pH 3.2, 0.01 M)-acetonitrile-methanol (30:50:20, v/v/v). The developed method has a short run time of 12 min with a flow rate of 1.0 ml/min. The injector volume was set to 20 microl and acquisition was carried out using a PDA detector while processing was done by timed wavelength extraction. The percentage R.S.D. and recovery in all studies indicated that the method was suitable for the intended purpose. The validated method was found to be linear and precise in the working range. Suitability of storage at cold temperature was ensured along with complete sample recovery.